The mass spectrometry (MS) core will generate global profiles of protein levels, characterize post-translational modifications (PTMs), define chemical structures of small biological molecules like lipopolysaccharides (LPS), integrate data from global protein and mRNA profiles to produce new hypotheses for testing, develop software for analysis of disparate global data sets and refine analytical methods for analysis of samples of limited availability and protein PTM analysis. Thus the purpose of the MS core is to be a mass spectrometry based resource for our biodefense collaborators interested in qualitative and quantitative proteomic analyses, determining protein primary and secondary structures as well as the structure of small biomolecules and integration of mRNA profiling and proteomic protein expression experiments. Specific Aim 1. Global analysis of protein regulation. We will determine changes in proteome expression resulting from select biochemical/environmental perturbations as well as genetic differences between strains. This will be done using multidimensional chromatography (MDLC) separation of peptides and both qualitative and quantitative (i.e. isotope coded affinity tags or ICAT) mass spectrometry (MS). To enable more rapid and thorough measurement of quantitative changes to proteomes, we will construct microbial-specific annotated peptide databases (APD). We will develop/refine methods for more sensitive analysis of samples of limited availability such as bacterial infected macrophages. Specific Aim 2. Structural characterization of biomolecules. We will provide basic support for determination of protein post-translational modifications (PTMs) and structural analysis of small biomolecules like lipopolysaccharides (LPS). For these experiments we will use both low energy collision induced dissociation (CID) on an electrospray ionization quadrupole time-of-flight (ESI-QTOF) MS and electron capture dissociation (ECD) on Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS). We will develop and implement novel PTM-tagging chemistries. Specific Aim 3. Computational support for data integration. We will use novel tools developed at the Institute for Systems Biology (ISB) to integrate and interpret data with the goal of generating new hypotheses for testing. Protein regulatory networks and mRNA profiling will be analyzed separately and together. First round results will be used to design new experiments that will refine our understanding of microbial biochemistry. We will 1) start with perturbations based on current knowledge of regulatory networks, 2) re-evaluate the regulatory network in light of new data and 3) derive additional new perturbations for next round global analyses. We will develop and visualize computational models that integrate mRNA and protein expression data with the host-microbe protein regulatory network scaffolds to generate predictive models of host- microbe network states.